CXCL13 is a predictive biomarker in idiopathic multicentric Castleman disease

Idiopathic multicentric Castleman disease (iMCD) is a rare and poorly-understood cytokine storm-driven inflammatory disorder. Interleukin-6 (IL-6) is a known disease driver in some patients, but anti-IL-6 therapy with siltuximab is not effective in all patients, and biomarkers indicating success at an early time point following treatment initiation are lacking. Here we show, by comparison of levels of 1,178 proteins in sera of healthy participants (N = 42), patients with iMCD (N = 88), and with related diseases (N = 60), a comprehensive landscape of candidate disease mediators and predictors of siltuximab response. C-X-C Motif Chemokine Ligand-13 (CXCL13) is identified and validated as the protein most prominently up-regulated in iMCD. Early and significant decrease in CXCL13 levels clearly distinguishes siltuximab responders from non-responders; a 17% reduction by day 8 following siltuximab therapy initiation is predictive of response at later time points. Our study thus suggests that CXCL13 is a predictive biomarker of response to siltuximab in iMCD.

Sex of participants was determined based on self-report. Sex was included as a covariate in analyses. Gender was not collected.
Samples were collected from a population of patients with idiopathic multicentric Castleman disease (iMCD). Primary Cohort: The majority of iMCD patient samples collected came from patients in the phase II clinical trial (NCT01024036), whose characteristics have been published. Additional iMCD samples were collected from patients with a more severe phenotype in order to represent patients across the iMCD spectrum. Population characteristics are described in Table S3. Related disease samples were collected from patients with diseases that have overlapping characteristics, including HHV8+MCD, hodgkin lymphoma, and rheumatoid arthritis. Validation Cohort: All iMCD patient samples collected came from patients in the phase I clinical trial (NCT00412321), whose characteristics have been published. Population characteristics are described in Table S3. IHC Cohort: All iMCD patient samples and samples from patients with reactive lymph nodes came from patients enrolled in the ACCELERATE Natural History Registry and represent patients who have been diagnosed with iMCD by at least one pathologist. Population characteristics are described in Table S3.
Primary Cohort: The primary cohort resulted from samples collected from 98 iMCD patients, 20 rheumatoid arthritis (RA) patients, 20 Hodgkin lymphoma (HL) patients, 20 HHV8-associated multicentric Castleman disease (HHV8+MCD) patients, and 44 healthy donors. Of the 98 iMCD patients, 79 were initially recruited for the phase II clinical trial for siltuximab (NCT01024036) and provided longitudinal samples with consent for use in future studies. These patients were recruited into the clinical trial from 38 hospitals in 19 countries. An additional 19 patient iMCD samples, as well as the 60 related disease samples (RA, HL, and HHV8+MCD) and 44 healthy donor samples were collected from sample banks housed by collaborators located in Japan, Norway, the United Kingdom and 3 sites (University of Arkansas for Medical Sciences, Brigham and Women's Hospital, and University of Pennsylvania) in the United States. Healthy donor samples were collected from individuals with no known disorders who were roughly age and sex matched to the iMCD patient population. Healthy donors were primarily recruited from faculty and staff at the collaborating institutions who self-reported having no known disorders. Self-selection bias for healthy donors may be present ('healthy donor effect'). Specifically, individuals who opted in as a healthy donor volunteer may be more likely to have lower rates of morbidity rates and more active lifestyle compared to the general population (e.g. active in the workforce). It is possible that this may bias the comparison of iMCD patients to a general healthy population.
Validation Cohort: The validation cohort resulted from 25 iMCD patient samples collected from the phase I siltuximab trial (NCT00412321). The phase I trial recruited patients with Castleman disease, multiple myeloma, and non-Hodgkin lymphoma from 9 centers in the United States. Of the 67 patients treated in the trial, 37 patients were diagnosed with Castleman disease, and 25 of them had iMCD and provided a sample for proteomics research.
Immunohistochemistry Cohort: The immunohistochemistry cohort consisted of iMCD patient lymph node samples collected from patients enrolled in the ACCELERATE Natural History Registry (NCT02817997). Patients are recruited into ACCELERATE through the Castleman Disease Collaborative Network and physician outreach. Patients self-enroll into ACCELERATE, which may result in self-selection bias and is likely to include a population of patients with more severe disease. A panel of clinicians and pathologists review each case and confirm the accuracy of iMCD diagnosis.

March 2021
Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size

Data exclusions
Replication Randomization Blinding Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
Sample size calculation was not performed as no given hypothesis was tested. The intention of this study was to perform biomarker discovery and to search for alternative therapeutic targets. To that end, we assembled the largest feasible cohort of iMCD patient samples then available. To do so, we partnered with Janssen Pharmaceuticals to obtain longitudinal patient samples collected from 79 patients in the phase II siltuximab trial (NCT01024036), which was a large enough sample size to demonstrate differences between the siltuximab and placebo arms based on intention to treat and estimated effect size. To enhance this sample size for the present study, we sought additional samples from collaborators around the world. The total number of real-world iMCD samples collected (N=19) was based on available banked patient samples for this rare disease. At the time, no accessible biobank existed and available biosamples were limited given the rarity of the disease and wide geographic distribution of patients. Related disease sample sizes were based on feasibility of collecting samples with associated clinical data. Healthy control sample size was based on consultation with the SomaLogic proteomic discovery assay developers, with the recommendation to collect approximately 1 healthy for every 5 disease state samples. Additional healthy donors were sought until assay initiation.
To validate our findings from the primary cohort, we assembled two additional independent cohorts. The validation cohort included 25 iMCD patients who participated in the phase I siltuximab trial (NCT00412321) and provided longitudinal biosamples for proteomics research. This was the largest known independent validation cohort with available longitudinal samples systematically collected pre-and post-treatment with siltuximab.
Lastly, to investigate CXCL13 in the lymph node, lymph node samples were collected from 19 iMCD patients, 11 RA patients, and 18 reactive lymph nodes. iMCD and reactive lymph node sample sizes were based on 80% power and 5% two-sided level of significance to detect the effect size observed in a pilot study of 6 iMCD and 5 sentinal lymph nodes (Pierson et al., Am J Hematol, 2018). 11 RA samples were collected as a positive control and was based on availability from the sample bank.
Outlier detection methods were established prior to analysis in order to identify samples that diverge from the overall population. Outliers were detected by aggregating the results of three conventional methods. Outliers were flagged if identified by 2 or more methods and were recommended for removal. Two samples from the primary cohort and from the validation cohort were found to be outliers based on at least 2 of these 3 methods: principal component analysis reconstruction residual, average pairwise distance (APW), and the APW to the K-nearest neighbors. These were excluded from further analysis.
Results obtained with the primary cohort were validated with an independent cohort of samples (validation cohort) obtained from the phase I siltuximab clinical trial (NCT00412321). Additionally, to validate the results found by the multiplex aptamer assay, which provides relative quantification, we quantified levels of CXCL13 in a subset of available serum samples from iMCD, RA, HHV8-MCD, and healthy donors in the Primary cohort by an enzyme-linked immunosorbent assay (ELISA) assay and found a strong correlation results. Validation of results are reported in the manuscript.
This study involved proteomic quantification of previously collected and banked biospecimens and therefore randomization is not applicable to the present analysis.
A large proportion of biosamples collected for the primary cohort in this study were collected during the phase II clinical trial for siltuximab (NCT01024036). As part of that study design, patients were block randomized (block size 6) with a computer generated randomisation schedule in a ratio of 2 treatment to 1 control.
This study involved proteomic quantification of previously collected and banked biospecimens and therefore blinding is not applicable to the present analysis. Technicians performing the proteomic quantification were however masked to sample identification.
This study includes a secondary analysis of samples collected as part of the phase II trial. As part of that study design, patients and investigators giving treatment were masked to allocation until protocol-defined failure, and investigators and independent assessors who evaluated outcomes were masked to allocation.